Development of a novel triplex-PCR assay for the identification of feline hemoplasma species and survey of hemoplasma species in cats in Türkiye

Three hemoplasma species, Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum, and Candidatus Mycoplasma turicensis, have been identified in domestic and wild felids. M. haemofelis may cause severe clinical manifestations in domestic cats, whereas others can lead to mild infections. Identification of these pathogens is done using molecular diagnostic tools like conventional-PCR or real-time PCR. However, these have disadvantages, such as the failure to differentiate species or high cost. This study aimed to develop a triplex-PCR method for the diagnosis and discrimination of feline hemoplasma species. Furthermore, it is aimed at providing molecular data on the epidemiology of feline hemoplasma species in Türkiye, where there is a paucity of information on these pathogens. Triplex-PCR primers amplifying the 16S rRNA gene regions of M. haemofelis (1022 bp), Ca. Mycoplasma haemominutum (607 bp), and Ca. Mycoplasma turicensis (456 bp) species were designed and optimized. Moreover, the detection limits of the method were also determined and it was found that the primers could detect 0.001 ng/μL amount of DNA for M. haemofelis, 0.0001 ng/μL for Ca. Mycoplasma haemominutum, and 0.0002 ng/μL for Ca. Mycoplasma turicensis in the sample. 286 cat blood samples obtained from Sivas province were researched for feline hemoplasma species. Feline hemoplasma species were detected in samples of 29 out of 286 cats (10.23 %). Five samples (1.74 %) were infected with only M. haemofelis, whereas 22 (7.69 %) were only infected with Ca. Mycoplasma haemominutum. Two samples (0.69 %) were found to be infected with both M. haemofelis and Ca. Mycoplasma haemominutum. Ca. Mycoplasma turicensis was not detected in this study. A triplex-PCR method that can be used for the identification and species differentiation of feline hemoplasma species in hosts was developed. Moreover, hemoplasma species were found to be circulating in cats in the study area, and it is recommended that veterinarians and animal owners take the necessary precautions to protect themselves and their cats from these pathogens.